Occlusions of blood vessels by intravascular clots cause or/and contribute to the pathogenesis of a variety of disease conditions including myocardial infarction, stroke and pulmonary embolism and thus represent a significant medical problem. Although fibrinolytics, such as plasminogen activators, have recently been used in the treatment of some of these diseases or conditions, their effectiveness and safety are still of great concern, especially under specific prothrombotic conditions such as deep vein thrombosis and pulmonary embolism.
Pulmonary thromboembolism, a leading cause of mortality, is most often a complication of deep venous thrombosis. Statistics show that more than 95% of pulmonary emboli result from thrombi in the deep venous system of the lower extremities. Despite advances in medicine, the incidence and/or recognition of embolism and deep vein thrombosis appears to be increasing. This increase has been attributed to higher survival of trauma patients, an increase in orthopedic surgeries for joint replacement, and the widespread use of indwelling catheters, as well as the overall increase in medical and surgical procedures, particularly in older patients. As a result, methods of preventing and treating deep vein thrombosis are required to reduce the incidence of pulmonary embolisms.
Factors which promote deep vein thrombosis were defined as early as the nineteenth century and include stasis, abnormalities of the blood vessel wall, and alterations in the blood coagulation system. The highest risk groups for deep vein thrombosis are surgical patients requiring 30 minutes or more of general anesthesia, postpartum patients, patients with right and left ventricular failure, patients with fractures or injuries involving the lower extremities, patients with chronic deep venous insufficiency of the legs, is patients on prolonged bed rest, cancer patients, obese individuals, and patients using estrogens. Treatment of deep vein thrombosis most often involves use of an anticoagulant such as heparin. Even with this well-known drug, however, there is no consensus regarding the optimum regimen of anticoagulant therapy that affords both safety and efficacy. In addition to anticoagulant therapy, thrombolytic agents, such as streptokinase and urokinase, have been used in the management of acute deep vein thrombosis.
Acute vascular occlusion by fibrin thrombi is also a common and dangerous complication of surgery. Development of venous (e.g., pulmonary embolism) and arterial (e.g., arterial re-occlusion) thrombi that may embolize vitally important blood vessels increases peri-operative morbidity and mortality. Cardiopulmonary bypass surgery, transcutaneous coronary angioplasty and carotid endarterectomy and other interventions may be complicated by formation of small blood clots, which embolize to cerebral vessels and cause serious persistent neurological disorders.
In contrast to settings where ischemia is the sole threat to health (e.g., acute myocardial infarction, AMI), the management of thrombosis in the surgical settings is complicated by the added risk of bleeding at and around the wound site imparted by anti-thrombotic therapy itself. Also, in contrast with AMI, where lysis of the mature clot occluding a single vessel is a primary goal for the fibrinolytic intervention (therapy), the goal of the post-surgical anti-thrombotic management is to maintain an adequate blood flow in diverse vascular areas at risk of occlusion by post-surgical clots (prophylaxis). In the surgical settings, the ideal thromboprophylactic agent effectively dissolves nascent pathological thrombi developed in a post-operative and recovery periods, while not dissolving hemostatic fibrin clots formed during and immediately after the operation.
In addition to the objective inadequacies of existing agents (see below), widespread use of thromboprophylaxis in surgical settings continues to receive limited enthusiasm for additional reasons. The clinical significance of silent thrombotic events (pulmonary and brain micro-embolism) is under-appreciated because the symptoms may be delayed in onset and attributed to other causes, while significance of bleeding is acutely evident in the surgical practice and frequently blamed on thromboprophylaxis.
Current approaches afford some measure of protection, but all have a limited benefit/risk ratio in the surgical setting. Mechanical filters trap large clots, but cannot be used to prevent arterial re-occlusion or cerebral micro-emboli. Anticoagulants (e.g. low molecular weight heparin) reduce the incidence of thrombosis in some vascular beds, but dosing is limited by the risk of post-operative bleeding. Heparin has been reported to increase the incidence of wound hematomas, which in turn may predispose to wound infection and dehiscence. Aspirin and anti-GPIIb/IIIa agents suppress platelet aggregation, but cannot prevent formation of fibrin monomers and clot propagation. In addition to serious side effects (in part discussed above), each particular anticoagulant agent inhibits one specific mechanism for formation of clots (e.g., activation of platelets or fibrin formation), but none affords 100% fidelity of anti-thrombotic protection. A modality to dissolve nascent pathological thrombi, formed even despite anti-coagulation or due to its inapplicability, is acutely needed.
Plasminogen activators (PA's) are effective thrombolytics, which could be theoretically used for this purpose, but they have fundamental inadequacies for thromboprophylactic applications including rapid blood clearance and inactivation, non-selective degradation of the extracellular matrix and hemostatic fibrin plugs. The risk of hemorrhage practically precludes their use in the surgical settings. No existing anti-thrombotic strategy permits selective dissolution of the nascent clots without affecting pre-existing hemostatic fibrin plugs.
Streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, and urokinase are members of a family of agents known as plasminogen activators. These compounds act to dissolve intravascular clots by activating plasmin, a protease that digests fibrin. Plasminogen, the inactive precursor of plasmin, is converted to plasmin by cleavage of a single peptide bond. Plasmin itself is a nonspecific protease that digests fibrin clots as well as other plasma proteins, including several coagulation factors.
Fibrinolytic therapy with plasminogen activators have been shown to be useful in the treatment of myocardial infarction and stroke. However, application of these agents to dissolution of clots formed or lodged in other vascular areas such as deep venous areas is limited by extremely rapid elimination and inactivation after bolus dosing (Plow, E. et al. 1995. FASEB J. 9:939–945; Narita, M. et al. 1995. J. Clin. Invest. 96:1164–1168). Both tPA and urokinase undergo rapid inactivation by a circulating plasminogen activator inhibitor and plasmin itself is inactivated by a circulating glycoprotein, α-2-antiplasmin (Collen, D. 1996. Circulation 93:857–865; Reilly, C. et al. 1991. Arterioscl. Thromb. 11:1276–1286). α-2-antiplasmin inactivates staphylokinase, while streptokinase is more resistant to this endogenous glycoprotein inhibitor (Collen, B. et al. 1993. Eur. J. Biochem. 216:307–314). Although therapeutic doses of plasminogen activators can overwhelm the potential inhibitory activity of plasminogen activator inhibitor and α-2-antiplasmin, other inhibitors of plasminogen activators also are present (C1-inhibitor, α-2-macroglobulin, anti-trypsin) and contribute to the decrease over time in the fibrinolytic response upon treatment with plasminogen activators (Collen, D. 1996. Circulation 93:857–865). Such inactivation, or degradation of plasminogen activators and plasmin reduce the effectiveness of thrombolytic therapy and thus fail to prevent re-occlusion of blood vessels.
To overcome this problem, attempts have been made to infuse plasminogen activators intravenously for prolonged periods of time with little success; failure was attributed to the harmful side effects such as bleeding, dissolution of hemostatic clots as well as nascent clots causing deleterious consequences, and uncontrolled tissue proteolysis that occurred after extravascular deposition of plasminogen activators.
Accordingly, several different approaches have been attempted to improve efficacy of these agents in deep vein thrombosis including: prolongation of the half-life of plasminogen activators in blood; protection of plasminogen activators from inactivation by inhibitors; and targeting plasminogen activators to fibrin and thrombi. For example, chemical modifications and incorporation of plasminogen activators into liposomes have been used to prolong the half-life of plasminogen activators in the circulation (Kajihara, J. et al. 1994. Biochim. Biophys. Acta 1199:202–208; Heeremans, J. et al. 1995. Thromb. Haemost. 73:488–494). However, these studies have shown that the activity of liposome-encapsulated plasminogen activators is strongly compromised by steric limitations. Genetically engineered tPA compounds have also been produced which possess altered pharmacokinetic properties, enhanced resistance to inhibitors, and higher fibrinolytic potency (Collen, D. 1996. Circulation 93:857–865; Collen, D. 1993. Lancet 342:34–36; Krishnamurti, C. et al. 1996. Blood 87:14–19; Lijnen, R. and D. Collen. 1992. Ann. NY Acad. Sci. 667:357–364). Several laboratories have explored conjugation of plasminogen activators with antibodies recognizing fibrin or activated platelets in order to localize plasmin generation to the clot (Holvoet, P. et al. 1993. Circulation 87:1007–1016; Runge, M. et al. 1996. Circulation 94:1412–1422; Fears, R. and G. Poste. 1994. Fibrinolysis 8:203–213). However, such conjugated plasminogen activators with affinity for clot components only bind to the superficial layer of the clot and do not enter into the clot interior (Sakharov, D. and D. Rijken. 1995. Circulation 92:1883–1890). In addition, clots bind only a small fraction of injected “fibrin-specific” plasminogen activator because of limited surface area of the formed clots. Such targeting also fails to distinguish nascent clots with deleterious consequences from pre-existing desired hemostatis clots.
Further, to date, none of these methods for modifying plasminogen activators prevent deposition of plasminogen activators in tissues, which can lead to an increase in harmful side effects; they all represent molecules or molecular complexes with sizes that do not exceed that of blood proteins. Such deposition leads to plasmin activation in tissues. Activated plasmin degrades the extracellular matrix, thus causing vascular remodeling, abnormal elevation of vascular permeability and even partial denudation of subendothelium (Plow et al. 1995. FASEB J. 9:939–945; Shreiber et al. 1995. J. Cell. Physiol. 165:107–118).
Accordingly, there is a need for methods of modifying plasminogen activators which not only decrease the rate of elimination and degradation of the plasminogen activators, but also prevent deposition of the plasminogen activator in the extrasvascular tissues. Further, compositions and methods are needed which selectively target nascent clots from within, while having minimal effects on pre-existing hemostatic clots.
Red blood cells (RBCs) normally have a life span of 120 days and thus can serve as natural carriers for drugs and biomolecules. Autologous RBCs can be easily obtained from the patient's blood, loaded with drug, and re-injected. RBCs have been used as carriers for drugs loaded into the inner volume of RBCs (Poznansky, M. and R. Juliano. 1984. Pharmacol. Rev. 36:277–324; Kirch, M. et al. 1994. Biotechnol. App. Biochem. 19:331–363; Kinoshita, K. and T. Tsong. 1978. Nature 272:258–260). In addition, methods for conjugation of proteins to RBCs have been developed, including methods using a streptavidin-biotin pair as a cross-linker.
Streptavidin is a 60 kDa protein that possesses four high affinity biotin binding sites and the streptavidin-biotin pair is widely used in biomedicine as a cross-linking agent (Wilchek, M. and E. Bayer. 1988. Anal. Biochem. 171:1–32). Several groups have reported application of streptavidin-biotin technology in vivo for gamma-immunoscintigraphy (Kalofonos, H. et al. 1990. J. Nucl. Med. 31:1791–1796) and drug targeting (Pardridge, W. et al. 1995. Proc. Natl. Acad. Sci. USA 92:5592–5596; Muzykantov, V. et al. 1996. Proc. Natl. Acad. Sci. USA 93:5213–5218). Moreover, streptavidin induces no known harmful reactions in animals or humans (Kalofonos, H. et al. 1990. J. Nucl. Med. 31:1791–1796). Biotinylation of RBCs can be accomplished in manner which has no effect on the life span and biocompatibility of these cells in vivo in animals (Susuki, T. and G. Dale. 1987. Blood 70:791–795; Muzykantov, V. et al. 1991. Blood 78:2611–2618).
Biotinylation of proteins, including plasminogen activators, without significant reduction of functional activity of the plasminogen activator has been described (Muzykantov, V. et al. 1986. Biochem. Biophys. Acta, 884:355–363; Muzykantov, V. et al. 1995. Anal. Biochem., 226:279–287; Muzykantov, V. et al. 1996. J. Pharm. Exp. Ther., 279:1026–1034). In in vitro studies, polyvalent conjugation of various biotinylated proteins such as antibodies, enzyme peroxidase and fibrinolytic streptokinase with streptavidin conjugated-biotinylated RBCs (SA/b-RBC) was performed and high functional activity of these proteins bound to SA/b-RBC in vitro was reported (Muzykantov, V. et al. 1985. FEBS Lett. 182:62–66; Muzykantov, V. et al. 1986. Biochim. Biophys. Acta 884:355–363; Muzykantov, V. et al. 1987. Am. J. Pathol. 128:226–234).
However, polyvalent conjugation of biotinylated proteins to b-RBCs via streptavidin cross-linker profoundly compromises the biocompatibility of the carrier RBC. Binding of streptavidin to b-RBC leads to elimination of homologous restriction of both classical and alternative pathways of the complement thereby causing lysis of SA/b-RBC in the plasma (Muzykantov, V. et al. 1991. Blood 78:2611–2618; Muzykantov, V. et al. 1992. Int. J. Artif. Organs 15:620–627; Muzykantov, V. et al. 1993. FEBS Lett. 318:108–112). Streptavidin-induced cross-linking and membrane redistribution of the complement inhibitors, DAF and CD59, in biotinylated RBC membrane represents the likely mechanism for complement activation and lysis (Muzykantov, V. et al. 1992. Biochim. Biophys. Acta 1107:119–125; Zaltzman, A. et al. 1995. Biochem. J. 305:651–656). In addition, fixation of C3b complement component has been shown to lead to an increased rate of elimination of SA/b-RBC from the bloodstream via hepatic and splenic uptake (Muzykantov, V. et al. 1992. Int. J. Artif. Organs 15:620–627; Muzykantov, V. et al. 1996. Anal. Biochem. 214:109–119). Accordingly, drugs polyvalently conjugated to an RBC carrier via streptavidin can not be delivered to their targets in vivo.
This lack of biocompatibility of SA/b-RBC carrier can be overcome through modifications of the conjugation method. For example, monovalent coupling of streptavidin to b-RBCs has been demonstrated to produce a serum-stable carrier SA/b-RBC capable of binding up to 105 molecules of a biotinylated model protein per RBC (Muzykantov, V. et al. 1991. Biochem. J. 273:393–397; Muzykantov, V. et al. 1992. Biochim. Biophys. Acta 1107:119–125; Muzykantov, V. et al. 1993. Anal. Biochem. 208:338–342; Muzykantov, V. and R. Taylor. 1994. Anal. Biochem. 223:142–148; Muzykantov, V. et al. 1996. Anal. Biochem. 214:109–119;). B-RBC carrier, monovalently conjugated with a model biotinylated protein (b-IgG) via streptavidin, circulated for at least a day as a stable complex after intravenous injection in animals, with no evidence of lysis or hepatic uptake (Muzykantov, V. et al. 1996. Anal. Biochem. 214:109–119). In these studies, it was also found that the half-life b-IgG monovalently conjugated with SA/b-RBCs significantly exceeded that of non-conjugated b-IgG (Muzykantov, V. et al. 1996. Anal. Biochem. 214:109–119).
As shown herein, monovalent conjugation of a biotinylated plasminogen activator to a SA/b-RBC carrier results in prolonged circulation of the plasminogen activator in the bloodstream and decreased deposition of the plasminogen activator in the tissues. Further, as shown herein, monovalent conjugation of biotinylated soluble receptor for urokinase plasminogen activator, (b-suPAr), and monovalent conjugation of biotinylated tissue plasminogen activator, (tPA), to a SA/b-RBC carrier results in prolonged circulation of these biotinylated plasminogen activators in the bloodstream in a form of the receptor/RBC complex (b-suPAr/SA/b-RBC complex) and the tPA/RBC complex, respectively. Moreover, as shown herein b-suPAr/SA/b-RBC complex retains its ability to bind single chain urokinase plasminogen activator (scuPA) even after prolonged circulation in the bloodstream and non-covalent binding of this fibrinolytic precursor (scuPA) to the receptor conjugated to RBC carrier (i.e., to b-suPAr/SA/b-RBC complex) leads to scuPA activation and greater resistance to plasma inhibitors and thus provides increased fibrinolytic activity on the clot itself. As also shown herein pulmonary vascular uptake of tPA is increased by crosslinking tPA to biotinylated RBC to form a tPA/RBC complex, with the increase seen to a level that exceeds the level of tPA/RBC complex in larger blood vessels. In addition, conjugation of tPA and suPAr with a monoclonal antibody against human CR1 is shown herein to promote specific coupling of fibrinolytically active anti-thrombotic agents to red blood cells, limiting the non-specific binding and tissue uptake of the complexes.
Further, as shown herein coupling of an anti-thrombotic agent to a carrier such as an RBC alters is fibrinolytic profile rendering the anti-thrombotic agent relatively impermeable to pre-formed (hemostatic) clots and extravascular tissues, thereby reducing the risk of bleeding, while prolonging its circulation and ability to be incorporated inside nascent intravascular clots, providing their fast lysis from within.
Accordingly, the present invention relates to compositions comprising a therapeutic agent, such as an anti-thrombotic agent, biocompatibly coupled to a carrier sized to inhibit penetration into existing clots, such as a red blood cell, and methods of using these compositions in the selective dissolution of nascent intravascular blood clots while avoiding lysis of pre-existing (hemostatic) clots.